ACS Medicinal Chemistry Letters
Letter
for strong agonism of 1 and analogues, is not addressed by
18, rationalizing the intermediate FXR activation efficacy of
this novel chemotype. Further optimization potential might
therefore be present in the tert-butylphenyl region of 18.
Supporting this assumption, molecular docking of the
extended morpholino derivative 14 (Figure S3) indicated
further binding site occupation and suggested a potential
polar contact of the morpholine oxygen to Tyr361 at the end
of the pocket, which also forms a hydrogen bond with the
natural ligand CDCA.
Furthermore, 18 and 24 were nontoxic in human hepatocytes
(HepG2) up to 100 μM concentration (Figure 4b).
To further validate the FXR modulator activity of 18 and
24 in a more physiological setting than the hybrid assay, we
probed their effects on the human FXR response element
from the promoter region of BSEP, which is regulated by the
human full-length FXR:RXR heterodimer (Figure 4c). FXR
agonist 18 robustly activated FXR:RXR (EC50 = 3 μM),
whereas 24 acted as an FXR antagonist in competition with 1
on the FXR:RXR heterodimer as well.
In conclusion, from the nonselective virtual screening hit 5
as a lead, we have systematically probed the SAR of a novel
FXR ligand chemotype that turned out to be tunable in its
activity type between agonism and antagonism. With the
central heterocycle and side chain saturation as switches, FXR
activators and antagonists are accessible that comprise
preferable characteristics compared with the reference FXR
ligand 1. In particular, FXR agonist 18 and antagonist 24
evolved as optimized descendants of 5 with considerable
potency and high selectivity. Both compounds modulated
FXR activity in two cellular settings and bound to the
recombinant FXR LBD, which orthogonally validated their
effects as FXR-mediated. The compounds establish a valuable
new FXR modulator scaffold to expand the collection of FXR
ligand chemotypes as tools and potentially experimental
drugs.
As the remaining structural feature of this FXR ligand
chemotype, we then addressed the SAR of the central
heterocyclic component (21−25; Table 3). Replacing the
2,5-dimethylpyrrole of the partial agonist 19 by a 4-
methyloxazole in 21 led to a loss of FXR agonism and
resulted in a weak antagonist (IC50 = 13.9 μM). 1,3,4-
Oxadiazole analogue 22 lacking the methyl decorations
revealed a submicromolar IC50 value for competitive FXR
antagonism. 1-Methyl-1H-pyrazole analogue 23 was less
potent but demonstrated remarkably enhanced antagonist
efficacy, and unsubstituted pyrazole derivative 24 was both
potent and effective in competitive FXR antagonism (Figure
3). Incorporation of a third nitrogen atom in 1H-1,2,3-
triazole derivative 25 retained strong FXR antagonism but
with lower potency than for pyrazole 24. To further confirm
this interesting SAR, we prepared and characterized 26
comprising the antagonism-driving pyrazole heterocycle of 24
and the agonism-favored acrylate motif of 18 (Table 4). 26
turned out to be a very weak FXR agonist (<5% activation at
30 μM) and did not counteract FXR activation by 1. Thus,
only two minor structural variationsreplacement of the
dimethylpyrrole by a pyrazole and reduction of the acrylate
motifgovern the activity type of the FXR ligand chemotype
between the potent FXR agonism of 18 and the pronounced
antagonist activity of 24.
ASSOCIATED CONTENT
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sı
* Supporting Information
The Supporting Information is available free of charge at
Figures S1−S3, synthetic procedures and analytical
characterization data for compounds 6−26 and
intermediates, methods for in vitro characterization,
and computational procedures (PDF)
As a control experiment, we repeated the hybrid reporter
gene assay but added 24 only 1 h before lysis and
luminescence measurement to cells treated with 1 for full
FXR activation (Figure 3c). In this setting, no effect of 24 on
the reporter activity was observed, in contrast to coincubation
of 24 and 1, which demonstrated a strong decrease in
reporter activity. Thus, the observed activity of 24 was FXR-
mediated and not an artifact of luciferase inhibition.
Moreover, 24 robustly antagonized CDCA-induced activation
of the human FXR:RXR heterodimer in HepG2 cells, as
observed with a full-length FXR reporter construct bearing
the human FXR response element from the promoter region
of the FXR target gene BSEP to govern reporter activity
(Figure 3d). ITC further confirmed direct interaction of 24
with the recombinant FXR LBD with submicromolar binding
affinity (Kd = 0.3 μM; Figure 3e), and profiling on related
nuclear receptors characterized 24 as a selective FXR
antagonist (Figure 3f).
From the systematic SAR elucidation of the new FXR
ligand chemotype, 18 and 24 evolved as the most active FXR
modulators, prompting us to further evaluate their potential
as tool compounds for pharmacology and as leads for
medicinal chemistry (Figure 4). The pyrrole- and pyrazole-
based FXR ligands exhibited remarkably higher stability
against microsomal degradation than the reference FXR
agonist 1 (Figure 4a) and were also superior in terms of
lipophilicity (18, alogP 4.84; 24, alogP 3.99; 1, alogP 7.41).26
AUTHOR INFORMATION
■
Corresponding Author
Daniel Merk − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, D-60438 Frankfurt,
Phone: +49 69 79829327; Email: merk@
Authors
̈
Moritz Helmstadter − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, D-60438 Frankfurt, Germany
Jan Vietor − Institute of Pharmaceutical Chemistry, Goethe
University Frankfurt, D-60438 Frankfurt, Germany
Jana Sommer − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, D-60438 Frankfurt, Germany
Simone Schierle − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, D-60438 Frankfurt, Germany
Sabine Willems − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, D-60438 Frankfurt, Germany
Astrid Kaiser − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, D-60438 Frankfurt, Germany
Jurema Schmidt − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, D-60438 Frankfurt, Germany
Complete contact information is available at:
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ACS Med. Chem. Lett. 2021, 12, 267−274